JP2002302444A - Inhibitor or repairing agent of skin disorder caused by drying - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a preparation capable of inhibiting skin disorder caused by drying. SOLUTION: This preparation contains ectoine capable of preventing the expression of filaggrin gene in a cultured human keratinocyte from being reduced by exposure to a vapor phase, as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to cosmetics and dermatological preparations, and more particularly to cosmetics and preparations for suppressing or repairing skin damage due to drying.

[0002]

2. Description of the Related Art Natural moisturizing factors (NM)
F) plays an important role (BlankI.HJI. Dermato
l., 18, 433 (1952), Blank IHJI. Derm
atol., 21, 259 (1953)). It has been reported that amino acids, which are the main components of NMF, are produced by the degradation of filaggrin protein derived from keratohyalin granules (Scott IR et al. Biochem Biophys. Act).
a., 719, 110-117 (1982), Horii I. e.
t al., J. Dermatol., 10, 25-33 (198
3)). Filaggrin is a protein consisting of 317 amino acids. Since it was revealed that the amino acid which is the main component of NMF is derived from filaggrin, research on the relationship between filaggrin and a disease state exhibiting dry skin has been advanced. In recent years, amino acids in the stratum corneum have been reduced in dry skin such as senile xeroderma and atopic disease (Horii I. et al., Br. J. Dermatol., 121, 5).
87-592 (1989), Tanaka M. et al., Br.
J. Dermatol., 139, 618-621 (199
8)) and decreased expression of filaggrin (TezukaT. Et al., Dermatology, 188, 21-24).
(1994), Seguchi T, et al., Arch. Dermatol. R
es. 288, 442-446, (1996)). It is well known that a dry environment causes skin troubles such as rough skin.

[0003] From such a background, FR27771
85-A1 proposes a composition for the treatment of wrinkles in which a substance stimulating filaggrin synthesis is combined with other active substances to favorably regulate the water retention capacity. However, there is no description about what kind of substance stimulates filaggrin synthesis. In addition, JP
Japanese Patent Application Publication No. 000-26272 discloses that a proteolytic product derived from white lupine [or Lupinus albus ] seed powder increases the mRNA of human keratinocyte filaggrin and increases the thickness of the stratum corneum. There is a statement that there is. In addition, WO99 / 4
7117 describes that a composition containing a vitamin B3 compound acts as a humectant, and increases the level of skin proteins selected from filaggrin, keratin and invocrine, and enhances the ability of water to adsorb or absorb into the stratum corneum. Have been.

[0004]

As described above, substances that increase the production of filaggrin and increase the thickness of the stratum corneum or enhance the ability to absorb moisture into the stratum corneum have been proposed. However, involved in the production of filaggrin,
Moreover, it would still be desirable to provide substances or compounds that are effective from a broader perspective in maintaining a healthy skin condition.

[0005]

Means for Solving the Problems The present inventors have studied the factors involved in drying and the water content of the stratum corneum in human keratinocytes. As a result, the present inventors have found that the level of filaggrin protein and the expression of the gene are reduced by drying. I found it.

Furthermore, it has been found that when a specific cultured human keratinocyte is exposed to the gas phase, the expression level of the filaggrin gene is significantly reduced, and this phenomenon is correlated with the above-mentioned skin injury due to drying. On the other hand, in a culture system using the cultured human keratinocytes, a specific substance capable of preventing a decrease in the expression level of the filaggrin gene due to gas phase exposure, ectoine, can actually suppress or repair the skin injury caused by drying. I found it.

The present invention is based on this finding.

Therefore, according to the present invention, there is provided a preparation for suppressing or repairing a decrease in expression of a filaggrin gene comprising ectoine as an active ingredient.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The ectoin which is an active ingredient of the preparation of the present invention has the following formula:

[0010]

Embedded image

Which is known as a compound that certain bacteria accumulate to regulate osmotic pressure. Ectoin can be used without regard to origin, production method or purity, as long as it is for the purpose of the present invention.

The preparation of the present invention may contain an active ingredient or an excipient used in cosmetics and / or dermatological preparations in addition to the above ectoine. Other active ingredients for the purpose of the present invention include betaines and polyols other than ectoine. Such betaines may be any compound as long as they do not adversely affect ectoine, and include betaine (also called glycine betaine) and its derivatives, and the corresponding sultaine (where the carboxyl of betaine has the carboxyl of betaine). (Replaced by sulfo). Typical of such derivatives are one to three of the betaine N-methyl groups interrupted by another optionally branched saturated or unsaturated hydrocarbon chain or an amide bond of the formula The hydrocarbon chain (wherein
m and n are independently an integer from 1 to 30)

[0013]

Embedded image

Or the length of the hydrocarbon chain between the quaternary ammonium group and the carboxyl or sulfo group varies (for example, when the number of carbon atoms is 2 to 2).
6) or a polymer having a plurality of betaine residues as pendant groups. Although not limited, specific examples of such a derivative include, in addition to those described above, γ-butyrobetaine, decylbetaine, laurylbetaine, myristylbetaine, cetylbetaine, stearylbetaine, behenylbetaine, lauramidepropylbetaine, oleamide. Amidopropyl betaine, palmitamide propyl betaine, γ-butyrobetaine, lauryl sultaine, cocos lutein, poly (methacryloyloxyethyl betaine), poly (methacryloyloxyethyl betaine-co-2-hydroxyethyl methacrylic acid) and the like. be able to.

On the other hand, examples of polyols include compounds having two or more hydroxyl groups per molecule and derivatives thereof, for example, ethylene oxide adducts of polyols and the like. Specific examples of such polyols include glycerin, 1,3-propanediol, 2-methyl-1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, diglycerin, erythritol Gluconic acid, 1,2,6-hexanetriol, inositol, lactitol, maltitol, mannitol, xylitol and the like.

In the preparation of the present invention, ectoin can be contained in an amount of 0.0001 to 20% by weight, preferably 0.001 to 0.1% by weight, based on the total weight of the preparation. In any case, based on the results obtained in accordance with the test method or evaluation method described below for each specific use mode, and further considering the results of actual use by volunteers and the like on a trial basis, it is possible to obtain specialized knowledge in the field of dermatology. Homes will readily be able to determine the optimal dosage for the active ingredients to work.

The preparation of the present invention refers to those applied to cosmetics, pharmaceuticals, quasi-drugs, and hulls. , Ointment, cream, water-oil two-layer system, water-oil-powder three-layer system, and the like. That is, basic cosmetics can be widely applied to various forms such as face wash, lotion, milky lotion, cream, gel, essence (serum), pack, mask and the like. If it is a makeup cosmetic, it can be widely applied to forms such as foundations. Furthermore, any drug or quasi-drug can be widely applied to various ointments and the like. The form and form of the external preparation for skin of the present invention are not limited to these forms and forms.

The components used to prepare the preparations can be selected according to their dosage form and form, and such components or additives include liquid fats, solid fats, waxes, esters and the like. Oil, hydrocarbon oil, silicone resin, silicone, anionic surfactant, anionic surfactant, cationic surfactant, amphoteric surfactant, nonionic surfactant, lower alcohol, sterols,
Water-soluble polymers, sequestering agents, neutralizing agents, pH adjusters, antibacterial agents, fragrances, dyes and the like can be mentioned.

The above-mentioned preparations can be applied directly to volunteers to confirm the efficacy. Prior to this, it is another aspect of the present invention to determine whether or not to suppress skin damage due to drying, which is the present invention. Can be confirmed by the method of evaluating the substance.

The human keratinocytes used in such an evaluation method can be any type of human keratinocytes as long as the amount of filaggrin protein and the variation in gene expression can be significantly discriminated by culturing in the presence and absence of a test substance, respectively. It may be a cell. However, preferably, human foreskin-derived normal cells and mouse-derived 3T3 cells (ATCC CRL-1658) as feeder layers (supporting cells)
A cultured cell line consisting of These cell lines are described, for example, in Rheinwald et al., Cell 6,
331-344 (1975).
After culturing T3 cells in a suitable Petri dish and treating with mitomycin C, thus forming a feeder cell layer,
Seeding normal keratinocytes on the layer, for example, 9
It can be prepared by culturing in a 5% air-5% carbon gas environment at 37 ° C. until it becomes confluent.

Usually, the system cultured in the absence of the preparation (control) is preferably treated in parallel with the culture in the presence of the preparation (evaluation system).

[0022]

EXAMPLES The present invention will be further described below with reference to specific examples, but these examples are not intended to limit the present invention. The percentages used in the description are by weight unless otherwise specified. Measurement test of filaggrin gene expression level: (1) Cultured cells Normal keratinocytes derived from human foreskin and 3T derived from mouse
Three cells were used. (2) Culture medium for cultured cells The culture medium contains DMEM-Ham's F12 (3: 1), hydrocortisone, cholera enterotoxin, epidermal growth factor, insulin and 10% FBS. (3) Culture Rheinwald et al., Cell 6, 331-344 (197)
Culture according to the method of 5).

The outline is shown below. 3T3 cells were treated with mitomycin C and used as feeder layers (feeder cells). Human normal keratinocytes 1 × 10
^Five cells were seeded on 3T3 cells treated with mitomycin C, and cultured at 37 ° C in a 95% air-5% carbon gas environment until they became confluent. Subsequently, gas phase exposure was performed by removing the culture supernatant. Each sample was added 30 minutes before exposure to the gas phase and cultured at 37 ° C. A sample to which no sample was added was used as a control. Gas phase exposure was performed at 37 ° C. for 0-10 hours. (4) Measurement of filaggrin protein amount After the sample was added to the culture system, protein was extracted from cells exposed to gas phase for a certain period of time. According to a conventional method, filaggrin protein or filaggrin protein precursor (profilaggrin) was quantified by Western blotting. The outline is shown below. Extracted protein was converted to SDS-
After electrophoresis by PAGE, the DNA was transferred to a PDVF membrane, and an antigen-antibody reaction was performed using a mouse monoclonal antibody (anti-HUMAN Filaggrin MAb) specific to filaggrin as a primary antibody. Then, add Horseradish p to the secondary antibody
An antigen-antibody reaction was performed using an antibody labeled with eroxidase or alkaline phosphatase. After the reaction, the measurement was carried out using a coloring method or a chemical coloring method in which an enzymatic reaction was performed using a chromogenic substrate corresponding to the labeling enzyme. The band was quantified using an NIH image (analysis program). (5) Measurement of Filaggrin Gene Expression Level After each test substance was added to the above culture system, RNA was extracted from cells exposed to gas phase for a certain period of time using the AGPC method. RT-PCR was performed on these RNAs as described below. Primers specific for filaggrin were added to McKinley-Gr
ant LJ et al., Proc. Natl. Acad. Sci. USA,
86, 4848-4852 (1989). RT-PC
For R, once mRNA was converted into cDNA with reverse transcriptase, and using that as a template, a polymerase reaction (known reaction) was carried out using the above primers to measure filaggrin mRNA. The PCR product was electrophoresed on an agarose gel, and the gel after electrophoresis was immersed in an ethidium bromide solution, and then the DNA was measured. The DNA band was quantified using NIH images (analysis program). Correction was performed using aldehyde glycer-3-phosphate dehydrogenase (G3PDH). (6) Results (6-1) FIG. 1 shows temporal changes in the amount of filaggrin protein when human keratinocytes were exposed to the gas phase.
From the figure, it is clear that the filaggrin protein of human keratinocytes is significantly reduced by drying. (6-2) FIG. 2 shows the change over time in the expression level of the filaggrin gene in human keratinocytes due to the gas phase exposure.
From the figure, it is clear that the expression of the filaggrin gene in human keratinocytes is significantly reduced by drying. Further, the decrease occurs within a relatively short time. (6-3) The effect of suppressing damage to the amount of filaggrin protein due to gas phase exposure (drying stimulus) in human keratinocytes was determined by the amount of filaggrin protein when ectoine was used as a sample to be added to the culture system and when it was not used. evaluated. The results are shown in FIG. (6-4) The effect of suppressing injury that reduces the expression of filaggrin gene due to gas-phase exposure (dry stimulation) in human keratinocytes was evaluated based on the expression level of filaggrin gene depending on whether ectoine was added to the culture system. FIG. 4 shows the results.

From these figures it can be seen that the preparation according to the invention is:
It is confirmed that the decrease in the water content of the stratum corneum caused by drying of human keratinocytes can be significantly suppressed, and the decrease in the protein amount and gene expression of filaggrin, which is a raw material of amino acids essential for maintaining the water content of the stratum corneum, can be significantly suppressed. Thus, when the preparation of the present invention is applied to the skin, a healthy skin condition is maintained even in a dry environment.

Hereinafter, formulation examples of the preparation according to the present invention will be shown. Formulation Example 1 Behenyl alcohol 1.0% Stearyl alcohol 2.0% Squalane 10.0% Pentaerythyl tetraoctanoate 5.0% Lanolin 5.0% Paraben 0.3% Polyoxyethylene behenyl alcohol 1.0% Glycerin 10. 0% Ectoin 0.1% Sodium acetyl hyaluronate 0.1% Purified water Remaining Formulation Example 2 Liquid paraffin 10.0% Octamethylcyclotetrasiloxane 5.0% 1,3-Butyl glycol 10.0% Glycerin 3.0 % Ascorbic acid derivative 3.0% ectoine 0.5% carboxyl vinyl polymer 0.15% xanthan gum 0.25% stearic acid 1.5% cetyl alcohol 0.5% polyoxyethylene (10) monooleate 1.0 % Stearic acid monoglyceride 1.0% Ethanol 3.0% Incense Appropriate amount Purified water Residue Formulation Example 3 Cetostearyl alcohol 3.5% Squalane 20.0% Beeswax 3.0% Lanolin 5.0% Paraben 0.3% Polyoxyethylene (20) sonobitan monopalmitate 2.0% Stearic acid monoglyceride 2.0% Ectoin 0.2% Vitamin A 2.0% Fragrance Appropriate amount Purified water Remaining Formulation Example 4 Talc 3.0% Titanium dioxide 5.0% Bengala 0.5% Yellow iron oxide 1.4% Black Iron oxide 1.0% Polyoxyethylene sorbitan monostearate 0.9% Triethanolamine 1.0% Propylene glycol 5.0% Ectoin 0.5% Stearic acid 2.2% Isohexadecyl alcohol 7.0% Stearin Acid monoglyceride 2.0% lanolin 2.0% paraffin 8.0% paraben qs perfume qs

[Brief description of the drawings]

FIG. 1 is a graph showing the time-dependent change in filaggrin protein level due to drying (gas phase exposure) of cultured human keratinocytes. The results are shown as mean ± SEM value in trials n = 4 to 5. Note that ** means p <0.01.

FIG. 2 is a graph showing changes in the expression level of the filaggrin gene over time due to drying (gas phase exposure) of cultured human keratinocytes. Mean ± S in trials n = 4-6
It is indicated by EM value. Note that ** means p <0.01, and * means p <0.05.

FIG. 3 is a graph showing the effect of ectoin on the decrease in filaggrin gene expression due to drying of cultured human keratinocytes. The results are shown by the average ± SEM value in trial n = 3. Note that ** is p <0.01.

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Claims (3)

[Claims] 1. A preparation for suppressing or repairing a decrease in the expression of a filaggrin gene comprising ectoine as an active ingredient. 2. The preparation according to claim 1, wherein the expression of the filaggrin gene is an event in human keratinocytes. 3. The preparation according to claim 1, wherein the decrease in the expression of the filaggrin gene is caused by drying of the skin.